Alpha-amylase mutants

ABSTRACT

The invention relates to a variant of a parent Termamyl-like α-amylase, which exhibits an alteration in at least one of the following properties relative to said parent α-amylase: i) improved pH stability at a pH from 8 to 10.5; and/or ii) improved Ca 2+  stability at pH 8 to 10.5, and/or iii) increased specific activity at temperatures from 10 to 60° C.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/665,667, filed Sep. 19, 2003, which is a divisional of U.S.application Ser. No. 09/769,864, filed on Jan. 25, 2001, which is adivisional of U.S. application Ser. No. 09/183,412, filed on Oct. 30,1998, and claims priority under 35 U.S.C. 119 of Danish application no.1240/97, filed on Oct. 30, 1997, Danish application no. PA 1998 00936,filed on Jul. 14, 1998, U.S. provisional application No. 60/064,662,filed on Nov. 6, 1997 and U.S. provisional application No. 60/093,234,filed on Jul. 17, 1998, the contents of which are fully incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates to variants (mutants) of parentTermamyl-like α-amylases with higher activity at medium temperaturesand/or high pH.

BACKGROUND OF THE INVENTION

α-Amylases (α-1,4-glucan-4-glucanohydrolases, EC 3.2.1.1) constitute agroup of enzymes which catalyze hydrolysis of starch and other linearand branched 1,4-glucosidic oligo- and polysaccharides.

There is a very extensive body of patent and scientific literaturerelating to this industrially very important class of enzymes. A numberof α-amylases such as Termamyl-like α-amylases variants are known frome.g. WO 90/11352, WO 95/10603, WO 95/26397, WO 96/23873 and WO 96/23874.

Among more recent disclosures relating to α-amylases, WO 96/23874provides three-dimensional, X-ray crystal structural data for aTermamyl-like α-amylase which consists of the 300 N-terminal amino acidresidues of the B. amyloliquefaciens α-amylase (BAN™) and amino acids301-483 of the C-terminal end of the B. licheniformis α-amylasecomprising the amino acid sequence (the latter being availablecommercially under the tradename Termamyl™), and which is thus closelyrelated to the industrially important Bacillus α-amylases (which in thepresent context are embraced within the meaning of the term“Termamyl-like α-amylases”, and which include, inter alia, the B.licheniformis, B. amyloliquefaciens (BAN™) and B. stearothermophilus(BSG™) α-amylases). WO 96/23874 further describes methodology fordesigning, on the basis of an analysis of the structure of a parentTermamyl-like α-amylase, variants of the parent Termamyl-like α-amylasewhich exhibit altered properties relative to the parent.

BRIEF DISCLOSURE OF THE INVENTION

The present invention relates to novel α-amylolytic variants (mutants)of a Termamyl-like α-amylase which exhibit improved wash performance(relative to the parent α-amaylase) at high pH and at a mediumtemperature.

The term “medium temperature” means in the context of the invention atemperature from 10° C. to 60° C., preferably 20° C. to 50° C.,especially 30-40° C.

The term “high pH” means the alkaline pH which today are used forwashing, more specifically from about pH 8 to 10.5.

In the context of the invention a “low temperature α-amylase” means anα-amylase which has an relative optimum activity in the temperaturerange from 0-30° C.

In the context of the invention a “medium temperature α-amylase” meansan α-amylase which has an optimum activity in the temperature range from30-60° C. For instance, SP690 and SP722 α-amaylases, respectively, are“medium temperature α-amylases.

In the context of the invention a “high temperature α-amylase” is anα-amylase having the optimum activity in the temperature range from60-110° C. For instance, Termamyl is a “high temperature α-amylase.

Alterations in properties which may be achieved in variants (mutants) ofthe invention are alterations in:

the stability of the Termamyl-like α-amylase at a pH from 8 to 10.5,and/or the Ca²⁺ stability at pH 8 to 10.5, and/or the specific activityat temperatures from 10 to 60° C., preferably 20-50° C., especially30-40° C.

It should be noted that the relative temperature optimum often isdependent on the specific pH used. In other words the relativetemperature optimum determined at, e.g., pH 8 may be substantiallydifferent from the relative temperature optimum determined at, e.g., pH10.

The Temperature's Influence on the Enzymatic Activity

The dynamics in the active site and surroundings are dependent on thetemperature and the amino acid composition and of strong importance forthe relative temperature optimum of an enzyme. By comparing the dynamicsof medium and high temperature α-amylases, regions of importance for thefunction of high temperature α-amylases at medium temperatures can bedetermined. The temperature activity profile of the SP722 α-amaylase(SEQ ID NO: 2) and the B. licheniformis α-amylase (available from NovoNordisk as Termamyl®) (SEQ ID NO: 4) are shown in FIG. 2.

The relative temperature optimum of SP722 in absolute activities areshown to be higher at medium range temperatures (30-60° C.) than thehomologous B. licheniformis α-amylase, which have an optimum activityaround 60-100° C. The profiles are mainly dependent on the temperaturestability and the dynamics of the active site residues and theirsurroundings. Further, the activity profiles are dependent on the pHused and the pKa of the active site residues.

In the first aspect the invention relates to a variant of a parentTermamyl-like α-amylase, which variant has α-amylase activity, saidvariant comprises one or more mutations corresponding to the followingmutations in the amino acid sequence shown in SEQ ID NO: 2:

T141, K142, F143, D144, F145, P146, G147, R148, G149, Q174, R181, G182,D183, G184, K185, A186, W189, S193, N195, H107, K108, G109, D166, W167,D168, Q169, S170, R171, Q172, F173, F267, W268, K269, N270, D271, L272,G273, A274, L275, K311, E346, K385, G456, N457, K458, P459, G460, T461,V462, T463.

A variant of the invention have one or more of the followingsubstitutions or deletions:

T141A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,W,Y,V;K142A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;F143A,D,R,N,C,E,Q,G,H,I,L,K,M,P,S,T,W,Y,V;D144A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;F145A,D,R,N,C,E,Q,G,H,I,L,K,M,P,S,T,W,Y,V;P146A,D,R,N,C,E,Q,G,H,I,L,K,M,F,S,T,W,Y,V;G147A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;R148A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;G149A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;R181*,A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;G182*,A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;D183*,A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;G184*,A,R,D,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;K185A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;A186D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;W189A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,Y,V;S193A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V;N195A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;H107A,D,R,N,C,E,Q,G,I,L,K,M,F,P,S,T,W,Y,V;K108A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;G109A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;D166A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;W167A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,Y,V;D168A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;Q169A,D,R,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;S170A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V;R171A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;Q172A,D,R,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;F173A,D,R,N,C,E,Q,G,H,I,L,K,M,P,S,T,W,Y,V;Q174*,A,D,R,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;F267A,D,R,N,C,E,Q,G,H,I,L,K,M,P,S,T,W,Y,V;W268A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,Y,V;K269A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;N270A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;D271A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;L272A,D,R,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V;G273A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;A274D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;L275A,D,R,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V;K311A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;E346A,D,R,N,C,Q,G,H,I,K,L,M,F,P,S,T,W,Y,V;K385A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;G456A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;N457A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;K458A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;P459A,D,R,N,C,E,Q,G,H,I,L,K,M,F,S,T,W,Y,V;G460A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;T461A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,W,Y,V;V462A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;T463A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,W,Y,V.

Preferred are variants having one or more of the following substitutionsor deletions:

K142R; S193P; N195F; K269R,Q; N270Y,R,D; K311R; E346Q; K385R; K458R;P459T; T461P; Q174*; R181Q,N,S; G182T,S,N; D183*; G184*;K185A,R,D,C,E,Q,G,H,I,L,M,N,F,P,S,T,W,Y,V; A186T,S,N,I,V,R; W189T,S,N,Q.

Especially preferred are variants having a deletion in positions D183and G184 and further one or more of the following substitutions ordeletions:

K142R; S193P; N195F; K269R,Q; N270Y,R,D; K311R; E346Q; K385R; K458R;P459T; T461P; Q174*; R181Q,N,S; G182T,S,N;K185A,R,D,C,E,Q,G,H,I,L,M,N,F,P,S,T,W,Y,V; A186T,S,N,I,V,R; W189T,S,N,Q.

The variants of the invention mentioned above exhibits an alteration inat least one of the following properties relative to the parentα-amylase:

-   i) improved pH stability at a pH from 8 to 10.5; and/or-   ii) improved Ca²⁺ stability at pH 8 to 10.5, and/or-   iii) increased specific activity at temperatures from 10 to 60° C.,    preferably 20-50° C., especially 30-40° C. Further, details will be    described below.

The invention further relates to DNA constructs encoding variants of theinvention; to methods for preparing variants of the invention; and tothe use of variants of the invention, alone or in combination with otherenzymes, in various industrial products or processes, e.g., indetergents or for starch liquefaction.

In a final aspect the invention relates to a method of providingα-amylases with altered pH optimum, and/or altered temperature optimum,and/or improved stability.

Nomenclature

In the present description and claims, the conventional one-letter andthree-letter codes for amino acid residues are used. For ease ofreference, α-amylase variants of the invention are described by use ofthe following nomenclature:

Original amino acid(s):position(s):substituted amino acid(s)

According to this nomenclature, for instance the substitution of alaninefor asparagine in position 30 is shown as:

-   -   Ala30Asn or A30N        a deletion of alanine in the same position is shown as:    -   Ala30* or A30*        and insertion of an additional amino acid residue, such as        lysine, is shown as:    -   Ala30AlaLys or A30AK

A deletion of a consecutive stretch of amino acid residues, such asamino acid residues 30-33, is indicated as (30-33)* or Δ(A30-N33).

Where a specific α-amylase contains a “deletion” in comparison withother α-amylases and an insertion is made in such a position this isindicated as:

-   -   *36Asp or *36D        for insertion of an aspartic acid in position 36        Multiple mutations are separated by plus signs, i.e.:    -   Ala30Asp+Glu34Ser or A30N+E34S        representing mutations in positions 30 and 34 substituting        alanine and glutamic acid for asparagine and serine,        respectively.

When one or more alternative amino acid residues may be inserted in agiven position it is indicated as

-   -   A30N,E or    -   A30N or A30E

Furthermore, when a position suitable for modification is identifiedherein without any specific modification being suggested, it is to beunderstood that any amino acid residue may be substituted for the aminoacid residue present in the position. Thus, for instance, when amodification of an alanine in position 30 is mentioned, but notspecified, it is to be understood that the alanine may be deleted orsubstituted for any other amino acid, i.e., any one of:

R,N,D,A,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is an alignment of the amino acid sequences of six parentTermamyl-like α-amylases. The numbers on the extreme left designate therespective amino acid sequences as follows:

-   1: SEQ ID NO: 2-   2: Kaoamyl-   3: SEQ ID NO: 1-   4: SEQ ID NO: 5-   5: SEQ ID NO: 4-   6: SEQ ID NO: 3.

FIG. 2 shows the temperature activity profile of SP722 (SEQ ID NO: 2)(at pH 9) and B. licheniformis α-amylase (SEQ ID NO: 4) (at pH 7.3).

FIG. 3 shows the temperature profile for SP690 (SEQ ID NO: 1), SP722(SEQ ID NO: 2), B. licheniformis α-amylase (SEQ ID NO: 4) at pH 10.

FIG. 4 is an alignment of the amino acid sequences of five α-amylases.The numbers on the extreme left designate the respective amino acidsequences as follows:

-   1: amyp_mouse-   2: amyp_rat-   3: amyp_pig porcine pancreatic alpha-amylase (PPA)-   4: amyp_human-   5: amy_altha A. haloplanctis alpha-amylase (AHA)

DETAILED DISCLOSURE OF THE INVENTION

The Termamyl-Like α-Amylase

It is well known that a number of α-amylases produced by Bacillus spp.are highly homologous on the amino acid level. For instance, the B.licheniformis α-amylase comprising the amino acid sequence shown in SEQID NO:. 4 (commercially available as Termamyl™) has been found to beabout 89% homologous with the B. amyloliquefaciens α-amylase comprisingthe amino acid sequence shown in SEQ ID NO: 5 and about 79% homologouswith the B. stearothermophilus α-amylase comprising the amino acidsequence shown in SEQ ID NO: 3. Further homologous α-amylases include anα-amylase derived from a strain of the Bacillus sp. NCIB 12289, NCIB12512, NCIB 12513 or DSM 9375, all of which are described in detail inWO 95/26397, and the α-amylase described by Tsukamoto et al.,Biochemical and Biophysical Research Communications, 151 (1988), pp.25-31, (see SEQ ID NO: 6).

Still further homologous α-amylases include the α-amylase produced bythe B. licheniformis strain described in EP 0252666 (ATCC 27811), andthe α-amylases identified in WO 91/00353 and WO 94/18314. Othercommercial Termamyl-like B. licheniformis α-amylases are comprised inthe products Optitherm™ and Takatherm™ (available from Solvay), Maxamyl™(available from Gist-brocades/Genencor), Spezym AA™ and Spezyme DeltaAA™ (available from Genencor), and Keistase™ (available from Daiwa).

Because of the substantial homology found between these α-amylases, theyare considered to belong to the same class of α-amylases, namely theclass of “Termamyl-like α-amylases”.

Accordingly, in the present context, the term “Termamyl-like α-amylase”is intended to indicate an α-amylase which, at the amino acid level,exhibits a substantial homology to Termamyl™, i.e., the B. licheniformisα-amylase having the amino acid sequence shown in SEQ ID NO: 4 herein.In other words, all the following α-amylases which has the amino acidsequences shown in SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7 or 8 herein, or theamino acid sequence shown in SEQ ID NO: 1 of WO 95/26397 (the same asthe amino acid sequence shown as SEQ ID NO: 7 herein) or in SEQ ID NO: 2of WO 95/26397 (the same as the amino acid sequence shown as SEQ ID NO:8 herein) or in Tsukamoto et al., 1988, (which amino acid sequence isshown in SEQ ID NO: 6 herein) are considered to be “Termamyl-likeα-amylase”. Other Termamyl-like α-amylases are α-amylases i) whichdisplays at least 60%, such as at least 70%, e.g., at least 75%, or atleast 80%, e.g., at least 85%, at least 90% or at least 95% homologywith at least one of said amino acid sequences shown in SEQ ID NOS: 1-8and/or ii) displays immunological cross-reactivity with an antibodyraised against at least one of said α-amylases, and/or iii) is encodedby a DNA sequence which hybridizes to the DNA sequences encoding theabove-specified α-amylases which are apparent from SEQ ID NOS: 9, 10,11, or 12 of the present application (which encoding sequences encodethe amino acid sequences shown in SEQ ID NOS: 1, 2, 3, 4 and 5 herein,respectively), from SEQ ID NO: 4 of WO 95/26397 (which DNA sequence,together with the stop codon TAA, is shown in SEQ ID NO: 13 herein andencodes the amino acid sequence shown in SEQ ID NO: 8 herein) and fromSEQ ID NO: 5 of WO 95/26397 (shown in SEQ ID NO: 14 herein),respectively.

In connection with property i), the “homology” may be determined by useof any conventional algorithm, preferably by use of the GAP progammefrom the GCG package version 7.3 (June 1993) using default values forGAP penalties, which is a GAP creation penalty of 3.0 and GAP extensionpenalty of 0.1, (Genetic Computer Group (1991) Programme Manual for theGCG Package, version 7, 575 Science Drive, Madison, Wis., USA 53711).

A structural alignment between Termamyl (SEQ ID NO: 4) and aTermamyl-like α-amylase may be used to identify equivalent/correspondingpositions in other Termamyl-like α-amylases. One method of obtainingsaid structural alignment is to use the Pile Up programme from the GCGpackage using default values of gap penalties, i.e., a gap creationpenalty of 3.0 and gap extension penalty of 0.1. Other structuralalignment methods include the hydrophobic cluster analysis (Gaboriaud etal., (1987), FEBS LETTERS 224, pp. 149-155) and reverse threading(Huber, T; Torda, AE, PROTEIN SCIENCE Vol. 7, No. 1 pp. 142-149 (1998).

Property ii) of the α-amylase, i.e., the immunological cross reactivity,may be assayed using an antibody raised against, or reactive with, atleast one epitope of the relevant Termamyl-like α-amylase. The antibody,which may either be monoclonal or poly-clonal, may be produced bymethods known in the art, e.g., as described by Hudson et al., PracticalImmunology, Third edition (1989), Blackwell Scientific Publications. Theimmunological cross-reactivity may be determined using assays known inthe art, examples of which are Western Blotting or radialimmunodiffusion assay, e.g., as described by Hudson et al., 1989. Inthis respect, immunological cross-reactivity between the α-amylaseshaving the amino acid sequences SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, or 8,respectively, has been found.

The oligonucleotide probe used in the characterisation of theTermamyl-like α-amylase in accordance with property iii) above maysuitably be prepared on the basis of the full or partial nucleotide oramino acid sequence of the α-amylase in question.

Suitable conditions for testing hybridisation involve pre-soaking in5×SSC and prehybridizing for 1 hour at −40° C. in a solution of 20%formamide, 5× Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and50 mg of denatured sonicated calf thymus DNA, followed by hybridisationin the same solution supplemented with 100 mM ATP for 18 hours at ˜40°C., followed by three times washing of the filter in 2×SSC, 0.2% SDS at40° C. for 30 minutes (low stringency), preferred at 50° C. (mediumstringency), more preferably at 65° C. (high stringency), even morepreferably at ˜75° C. (very high stringency). More details about thehybridisation method can be found in Sambrook et al., Molecular Cloning:A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989.

In the present context, “derived from” is intended not only to indicatean α-amylase produced or producible by a strain of the organism inquestion, but also an α-amylase encoded by a DNA sequence isolated fromsuch strain and produced in a host organism transformed with said DNAsequence. Finally, the term is intended to indicate an α-amylase whichis encoded by a DNA sequence of synthetic and/or cDNA origin and whichhas the identifying characteristics of the α-amylase in question. Theterm is also intended to indicate that the parent α-amylase may be avariant of a naturally occurring α-amylase, i.e. a variant which is theresult of a modification (insertion, substitution, deletion) of one ormore amino acid residues of the naturally occurring α-amylase.

Parent Hybrid α-Amylases

The parent α-amylase (i.e., backbone α-amylase) may be a hybridα-amylase, i.e., an α-amylase which comprises a combination of partialamino acid sequences derived from at least two α-amylases.

The parent hybrid α-amylase may be one which on the basis of amino acidhomology and/or immunological cross-reactivity and/or DNA hybridization(as defined above) can be determined to belong to the Termamyl-likeα-amylase family. In this case, the hybrid α-amylase is typicallycomposed of at least one part of a Termamyl-like α-amylase and part(s)of one or more other α-amylases selected from Termamyl-like α-amylasesor non-Termamyl-like α-amylases of microbial (bacterial or fungal)and/or mammalian origin.

Thus, the parent hybrid α-amylase may comprise a combination of partialamino acid sequences deriving from at least two Termamyl-likeα-amylases, or from at least one Termamyl-like and at least onenon-Termamyl-like bacterial α-amylase, or from at least oneTermamyl-like and at least one fungal α-amylase. The Termamyl-likeα-amylase from which a partial amino acid sequence derives may, e.g., beany of those specific Termamyl-like α-amylase referred to herein.

For instance, the parent α-amylase may comprise a C-terminal part of anα-amylase derived from a strain of B. licheniformis, and a N-terminalpart of an α-amylase derived from a strain of B. amyloliquefaciens orfrom a strain of B. stearothermophilus. For instance, the parentα-amylase may comprise at least 430 amino acid residues of theC-terminal part of the B. licheniformis α-amylase, and may, e.g.,comprise a) an amino acid segment corresponding to the 37 N-terminalamino acid residues of the B. amyloliquefaciens α-amylase having theamino acid sequence shown in SEQ ID NO: 5 and an amino acid segmentcorresponding to the 445 C-terminal amino acid residues of the B.licheniformis α-amylase having the amino acid sequence shown in SEQ IDNO: 4, or a hybrid Termamyl-like α-amylase being identical to theTermamyl sequence, i.e., the Bacillus licheniformis α-amylase shown inSEQ ID NO: 4, except that the N-terminal 35 amino acid residues (of themature protein) has been replaced by the N-terminal 33 residues of BAN(mature protein), i.e., the Bacillus amyloliquefaciens α-amylase shownin SEQ ID NO: 5; or b) an amino acid segment corresponding to the 68N-terminal amino acid residues of the B. stearothermophilus α-amylasehaving the amino acid sequence shown in SEQ ID NO: 3 and an amino acidsegment corresponding to the 415 C-terminal amino acid residues of theB. licheniformis α-amylase having the amino acid sequence shown in SEQID NO: 4.

Another suitable parent hybrid α-amylase is the one previously describedin WO 96/23874 (from Novo Nordisk) constituting the N-terminus of BAN,Bacillus amyloliquefaciens α-amylase (amino acids 1-300 of the matureprotein) and the C-terminus from Termamyl (amino acids 301-483 of themature protein). Increased activity was achieved by substituting one ormore of the following positions of the above hybrid α-amylase(BAN:1-300/Termamyl:301-483): Q360, F290, and N102. Particularlyinteresting substitutions are one or more of the followingsubstitutions: Q360E,D; F290A,C,D,E,G,H,I,K,L,M,N,P,Q,R,S,T; N102D,E;

The corresponding positions in the SP722 α-amylase shown in SEQ ID NO: 2are one or more of: S365, Y295, N106. Corresponding substitutions ofparticular interest in said α-amylase shown in SEQ ID NO: 2 are one ormore of: S365D,E; Y295 A,C,D,E,G,H,I,K,L,M,N,P,Q,R,S,T; and N106D,E.

The corresponding positions in the SP690 α-amylase shown in SEQ ID NO: 1are one or more of: S365, Y295, N106. The corresponding substitutions ofparticular interest are one or more of: S365D,E; Y295A,C,D,E,G,H,I,K,L,M,N,P,Q,R,S,T; N106D,E.

The above mentioned non-Termamyl-like α-amylase may, e.g., be a fungalα-amylase, a mammalian or a plant α-amylase or a bacterial α-amylase(different from a Termamyl-like α-amylase). Specific examples of suchα-amylases include the Aspergillus oryzae TAKA α-amylase, the A. nigeracid α-amylase, the Bacillus subtilis α-amylase, the porcine pancreaticα-amylase and a barley α-amylase. All of these α-amylases haveelucidated structures which are markedly different from the structure ofa typical Termamyl-like α-amylase as referred to herein.

The fungal α-amylases mentioned above, i.e., derived from A. niger andA. oryzae, are highly homologous on the amino acid level and generallyconsidered to belong to the same family of α-amylases. The fungalα-amylase derived from Aspergillus oryzae is commercially availableunder the tradename Fungamyl™.

Furthermore, when a particular variant of a Termamyl-like α-amylase(variant of the invention) is referred to—in a conventional manner—byreference to modification (e.g., deletion or substitution) of specificamino acid residues in the amino acid sequence of a specificTermamyl-like α-amylase, it is to be understood that variants of anotherTermamyl-like α-amylase modified in the equivalent position(s) (asdetermined from the best possible amino acid sequence alignment betweenthe respective amino acid sequences) are encompassed thereby.

In a preferred embodiment of the invention the α-amylase backbone isderived from B. licheniformis (as the parent Termamyl-like α-amylase),e.g., one of those referred to above, such as the B. licheniformisα-amylase having the amino acid sequence shown in SEQ ID NO: 4.

Altered Properties of Variants of the Invention

The following discusses the relationship between mutations which arepresent in variants of the invention, and desirable alterations inproperties (relative to those a parent Termamyl-like α-amylase) whichmay result therefrom.

Improved Stability at pH 8-10.5

In the context of the present invention, mutations (including amino acidsubstitutions) of importance with respect to achieving improvedstability at high pH (i.e., pH 8-10.5) include mutations correspondingto mutations in one or more of the following positions in SP722α-amylase (having the amino acid sequence shown in SEQ ID NO: 2): T141,K142, F143, D144, F145, P146, G147, R148, G149, R181, A186, S193, N195,K269, N270, K311, K458, P459, T461.

The variant of the invention have one or more of the followingsubstitutions (using the SEQ ID NO: 2 numbering):

T141A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,W,Y,V;K142A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;F143A,D,R,N,C,E,Q,G,H,I,L,K,M,P,S,T,W,Y,V;D144A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;F145A,D,R,N,C,E,Q,G,H,I,L,K,M,P,S,T,W,Y,V;P146A,D,R,N,C,E,Q,G,H,I,L,K,M,F,S,T,W,Y,V;G147A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;R148A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;G149A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;K181A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;A186D,R,N,C,E,Q,G,H,I,L,P,K,M,F,S,T,W,Y,V;S193A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V;N195A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;K269A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;N270A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;K311A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;K458A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;P459A,D,R,N,C,E,Q,G,H,I,L,K,M,F,S,T,W,Y,V;T461A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,W,Y,V.

Preferred high pH stability variants include one or more of thefollowing substitutions in the SP722 α-amylase (having the amino acidsequence shown in SEQ ID NO: 2):

K142R, R181S, A186T, S193P, N195F, K269R, N270Y, K311R, K458R, P459T andT461P.

In specific embodiments the Bacillus strain NCIB 12512 α-amylase havingthe sequence shown in SEQ ID NO: 1, or the B. stearothermophilusα-amylase having the sequence shown in SEQ ID NO: 3, or the B.licheniformis α-amylase having the sequence shown in SEQ ID NO: 4, orthe B. amyloliquefaciens α-amylase having the sequence shown in SEQ IDNO: 5 is used as the backbone, i.e., parent Termamyl-like α-amylase, forthese mutations.

As can been seen from the alignment in FIG. 1 the B. stearothermophilusα-amylase already has a Tyrosine at position corresponding to N270 inSP722. Further, the Bacillus strain NCIB 12512 α-amylase, the B.stearothermophilus α-amylase, the B. licheniformis α-amylase and the B.amyloliquefaciens α-amylase already have Arginine at positioncorresponding to K458 in SP722. Furthermore, the B. licheniformisα-amylase already has a Proline at position corresponding to T461 inSP722. Therefore, for said α-amylases these substitutions are notrelevant.

α-amylase variants with improved stability at high pH can be constructedby making substitutions in the regions found using the moleculardynamics simulation mentioned in Example 2. The simulation depicts theregion(s) that has a higher flexibility or mobility at high pH (i.e., pH8-10.5) when compared to medium pH. By using the structure of anybacterial alpha-amylase with homology (as defined below) to theTermamyl-like α-amylase (BA2), of which the 3D structure is disclosed inAppendix 1 of WO 96/23874 (from Novo Nordisk), it is possible tomodelbuild the structure of such alpha-amylase and to subject it tomolecular dynamics simulations. The homology of said bacterial α-amylasemay be at least 60%, preferably be more than 70%, more preferably morethan 80%, most preferably more than 90% homologous to the abovementioned Termamyl-like α-amylase (BA2), measured using the UWGCG GAPprogram from the GCG package version 7.3 (June 1993) using defaultvalues for GAP penalties [Genetic Computer Group (1991) Programme Manualfor the GCG Package, version 7, 575 Science Drive, Madison, Wis., USA53711]. Substitution of the unfavorable residue for another would beapplicable.

Improved Ca²⁺ Stability at pH 8-10.5

Improved Ca²⁺ stability means the stability of the enzyme under Ca²⁺depletion has been improved. In the context of the present invention,mutations (including amino acid substitutions) of importance withrespect to achieving improved Ca²⁺ stability at high pH include mutationor deletion in one or more positions corresponding to the followingpositions in the SP722 α-amylase having the amino acid sequence shown inSEQ ID NO: 2: R181, G182, D183, G184, K185, A186, W189, N195, N270,E346, K385, K458, P459.

A variant of the invention have one or more of the followingsubstitutions or deletions:

R181*,A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;G182*,A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;D183*,A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;G184*,A,R,D,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;K185A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;A186D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;W189A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,Y,V;N195A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;N270A,R,D,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;E346A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;K385A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;K458A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;P459A,R,D,N,C,E,Q,G,H,I,L,K,M,F,S,T,W,Y,V.

Preferred are variants having one or more of the following substitutionsor deletions:

R181Q,N; G182T,S,N; D183*; G184*;K185A,R,D,C,E,Q,G,H,I,L,M,N,F,P,S,T,W,Y,V; A186T,S,N,I,V; W189T,S,N,Q;N195F, N270R,D; E346Q; K385R; K458R; P459T.

In specific embodiments the Bacillus strain NCIB 12512 α-amylase havingthe sequence shown in SEQ ID NO: 1, or the B. amyloliquefaciensα-amylase having the sequence shown in SEQ ID NO: 5, or the B.licheniformis α-amylase having the sequence shown in SEQ ID NO: 4 areused as the backbone for these mutations.

As can been seen from the alignment in FIG. 1 the B. licheniformisα-amylase does not have the positions corresponding to D183 and G184 inSP722. Therefore for said α-amylases these deletions are not relevant.

In a preferred embodiment the variant is the Bacillus strain NCIB 12512α-amylase with deletions in D183 and G184 and further one of thefollowing substitutions: R181Q,N and/or G182T,S,N and/or D183*; G184*and/or

K185A,R,D,C,E,Q,G,H,I,L,M,N,F,P,S,T,W,Y,V and/or A186T,S,N,I,V and/orW189T,S,N,Q and/or N195F and/or N270R,D and/or E346Q and/or K385R and/orK458R and/or P459T.

Increased Specific Activity at Medium Temperature

In a further aspect of the present invention, important mutations withrespect to obtaining variants exhibiting increased specific activity attemperatures from 10-60° C., preferably 20-50° C., especially 30-40° C.,include mutations corresponding to one or more of the followingpositions in the SP722 α-amylase having the amino acid sequence shown inSEQ ID NO: 2:

H107, K108, G109, D166, W167, D168, Q169, S170, R171, Q172, F173, Q174,D183, G184, N195, F267, W268, K269, N270, D271, L272, G273, A274, L275,G456, N457, K458, P459, G460, T461, V462, T463.

The variant of the invention have one or more of the followingsubstitutions:

H107A,D,R,N,C,E,Q,G,I,L,K,M,F,P,S,T,W,Y,V;K108A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;G109A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;D166A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;W167A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,Y,V;D168A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;Q169A,D,R,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;S170A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V;R171A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;Q172A,D,R,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;F173A,D,R,N,C,E,Q,G,H,I,L,K,M,P,S,T,W,Y,V;Q174*,A,D,R,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;D183*,A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,W,Y,V;G184*,A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;N195A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;F267A,D,R,N,C,E,Q,G,H,I,L,K,M,P,S,T,W,Y,V;W268A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,Y,V;K269A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;N270A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;D271A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;L272A,D,R,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V;G273A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;A274D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;L275A,D,R,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V;G456A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;N457A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;K458A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;P459A,D,R,N,C,E,Q,G,H,I,L,K,M,F,S,T,W,Y,V;G460A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;T461A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,W,Y,V;V462A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;T463A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,W,Y,V.

Preferred variants has one or more of the following substitutions ordeletions: Q174*, D183*, G184*, K269S.

In a specific embodiment the B. licheniformis α-amylase having thesequence shown in SEQ ID NO: 4 is used as the backbone for thesemutations.

General Mutations in Variants of the Invention: Increased SpecificActivity at Medium Temperatures

The particularly interesting amino acid substitution are those thatincrease the mobility around the active site of the enzyme. This isaccomplished by changes that disrupt stabilizing interaction in thevicinity of the active site, i.e., within preferably 10 Å or 8 Å or 6 Åor 4 Å from any of the residues constituting the active site.

Examples are mutations that reduce the size of side chains, such as

-   Ala to Gly,-   Val to Ala or Gly,-   Ile or Leu to Val, Ala, or Gly-   Thr to Ser

Such mutations are expected to cause increased flexibility in the activesite region either by the introduction of cavities or by the structuralrearrangements that fill the space left by the mutation.

It may be preferred that a variant of the invention comprises one ormore modifications in addition to those outlined above. Thus, it may beadvantageous that one or more Proline residues present in the part ofthe α-amylase variant which is modified is/are replaced with anon-Proline residue which may be any of the possible, naturallyoccurring non-Proline residues, and which preferably is an Alanine,Glycine, Serine, Threonine, Valine or Leucine.

Analogously, it may be preferred that one or more Cysteine residuespresent among the amino acid residues with which the parent α-amylase ismodified is/are replaced with a non-Cysteine residue such as Serine,Alanine, Threonine, Glycine, Valine or Leucine.

Furthermore, a variant of the invention may—either as the onlymodification or in combination with any of the above outlinedmodifications—be modified so that one or more Asp and/or Glu present inan amino acid fragment corresponding to the amino acid fragment 185-209of SEQ ID NO: 4 is replaced by an Asn and/or Gln, respectively. Also ofinterest is the replacement, in the Termamyl-like α-amylase, of one ormore of the Lys residues present in an amino acid fragment correspondingto the amino acid fragment 185-209 of SEQ ID NO: 4 by an Arg.

It will be understood that the present invention encompasses variantsincorporating two or more of the above outlined modifications.

Furthermore, it may be advantageous to introduce point-mutations in anyof the variants described herein.

α-Amylase Variants Having Increased Mobility Around the Active Site:

The mobility of α-amylase variants of the invention may be increased byreplacing one or more amino acid residue at one or more positions closeto the substrate site. These positions are (using the SP722 α-amylase(SEQ ID NO: 2) numbering): V56, K108, D168, Q169, Q172, L201, K269,L272, L275, K446, P459.

Therefore, in an aspect the invention relates to variants being mutatedin one or more of the above mentioned positions.

Preferred substitutions are one or more of the following:

V56A,G,S,T; K108A,D,E,Q,G,H,I,L,M,N,S,T,V; D168A,G,I,V,N,S,T;Q169A,D,G,H,I,L,M,N,S,T,V; Q172A,D,G,H,I,L,M,N,S,T,V; L201A,G,I,V,S,T;K269A,D,E,Q,G,H,I,L,M,N,S,T,V; L272A,G,I,V,S,T; L275A,G,I,V,S,T;Y295A,D,E,Q,G,H,I,L,M,N,F,S,T,V; K446A,D,E,Q,G,H,I,L,M,N,S,T,V;P459A,G,I,L,S,T,V.

In specific embodiments of the invention the Bacillus strain NCIB 12512α-amylase having the sequence shown in SEQ ID NO: 1, or the B.stearothermophilus α-amylase having the sequence shown in SEQ ID NO: 3,or the B. licheniformis α-amylase having the sequence shown in SEQ IDNO: 4, or the B. amyloliquefaciens α-amylase having the sequence shownin SEQ ID NO: 5 are used as the backbone for these mutations.

As can been seen from the alignment in FIG. 1 the B. licheniformisα-amylase and the B. amyloliquefaciens α-amylase have a Glutamine atposition corresponding to K269 in SP722. Further, the B.stearothermophilus α-amylase has a Serine at position corresponding toK269 in SP722. Therefore, for said α-amylases these substitutions arenot relevant.

Furthermore, as can been seen from the alignment in FIG. 1 the B.amyloliquefaciens α-amylase has an Alanine at position corresponding toL272 in SP722, and the B. stearothermophilus α-amylase has a Isoleucineat the position corresponding to L272 in SP722. Therefore, for saidα-amylases these substitutions are not relevant.

As can been seen from the alignment in FIG. 1, the Bacillus strain 12512α-amylase has a Isoleucine at position corresponding to L275 in SP722.Therefore for said α-amylase this substitution is not relevant.

As can been seen from the alignment in FIG. 1 the B. amyloliquefaciensα-amylase has a Phenylalanine at position corresponding to Y295 inSP722. Further, the B. stearothermophilus α-amylase has an Asparagine atposition corresponding to Y295 in SP722. Therefore, for said α-amylasesthese substitutions are not relevant.

As can been seen from the alignment in FIG. 1 the B. licheniformisα-amylase and the B. amyloliquefaciens α-amylase have a Asparagine atposition corresponding to K446 in SP722. Further, the B.stearothermophilus α-amylase has a Histidine at position correspondingto K446 in SP722. Therefore, for said α-amylases these substitutions arenot relevant.

As can been seen from the alignment in FIG. 1 the B. licheniformisα-amylase, the B. amyloliquefaciens α-amylase and the B.stearothermophilus α-amylase have a Serine at position corresponding toP459 in SP722. Further, the Bacillus strain 12512 α-amylase has aThreonine at position corresponding to P459 in SP722. Therefore, forsaid α-amylases these substitutions are not relevant.

Stabilization of Enzymes Having High Activity at Medium Temperatures

In a further embodiment the invention relates to improving the stabilityof low temperature α-amylases (e.g, Alteromonas haloplanctis (Feller etal., (1994), Eur. J. Biochem 222:441-447), and medium temperatureα-amylases (e.g., SP722 and SP690) possessing medium temperatureactivity, i.e., commonly known as psychrophilic enzymes and mesophilicenzymes. The stability can for this particular enzyme class beunderstood either as thermostability or the stability at Calciumdepletion conditions.

Typically, enzymes displaying the high activity at medium temperaturesalso display severe problems under conditions that stress the enzyme,such as temperature or Calcium depletion.

Consequently, the objective is to provide enzymes that at the same timedisplay the desired high activity at medium temperatures without loosingtheir activity under slightly stressed conditions.

The activity of the stabilized variant measured at medium temperaturesshould preferably be between 100% or more and 50%, and more preferablybetween 100% or more and 70%, and most preferably between 100% or moreand 85% of the original activity at that specific temperature beforestabilization of the enzyme and the resulting enzyme should withstandlonger incubation at stressed condition than the wild type enzyme.

Contemplated enzymes include α-amylases of, e.g., bacterial or fungalorigin.

An example of such a low temerature α-amylase is the one isolated fromAlteromonas haloplanctis (Feller et al., (1994), Eur. J. Biochem222:441-447). The crystal structure of this alpha-amylase has beensolved (Aghajari et al., (1998), Protein Science 7:564-572).

The A. haloplanctis alpha-amylase (5 in alignment shown in FIG. 4) has ahomology of approximately 66% to porcine pancreatic alpha-amylase (PPA)(3 in the alignment shown in FIG. 4). The PPA 3D structure is known, andcan be obtained from Brookhaven database under the name 1OSE or 1DHK.Based on the homology to other more stable alpha amylases, stabilizationof “the low temperature highly active enzyme” from Alteromonashaloplanctis alpha-amylase, can be obtained and at the same timeretaining the desired high activity at medium temperatures.

FIG. 4 shown a multiple sequence alignments of five α-amylases,including the AHA and the PPA α-amylase. Specific mutations givingincreased stability in Alteromonas haloplantis alpha-amylase:

T66P, Q69P, R155P, Q177R, A205P, A232P, L243R, V295P, S315R.

Methods for Preparing α-Amylase Variants

Several methods for introducing mutations into genes are known in theart. After a brief discussion of the cloning of α-amylase-encoding DNAsequences, methods for generating mutations at specific sites within theα-amylase-encoding sequence will be discussed.

Cloning a DNA Sequence Encoding an α-AmylaseCloning a DNA SequenceEncoding an α-AmylaseCloning a DNA Sequence Encoding an α-AmylaseCloninga DNA Sequence Encoding an α-AmylaseCloning a DNA Sequence Encoding anα-AmylaseCloning a DNA Sequence Encoding an α-AmylaseCloning a DNASequence Encoding an α-AmylaseCloning a DNA Sequence Encoding anα-Amylase

The DNA sequence encoding a parent α-amylase may be isolated from anycell or microorganism producing the α-amylase in question, using variousmethods well known in the art. First, a genomic DNA and/or cDNA libraryshould be constructed using chromosomal DNA or messenger RNA from theorganism that produces the α-amylase to be studied. Then, if the aminoacid sequence of the α-amylase is known, homologous, labeledoligonucleotide probes may be synthesized and used to identifyα-amylase-encoding clones from a genomic library prepared from theorganism in question. Alternatively, a labeled oligonucleotide probecontaining sequences homologous to a known α-amylase gene could be usedas a probe to identify α-amylase-encoding clones, using hybridizationand washing conditions of lower stringency.

Yet another method for identifying α-amylase-encoding clones wouldinvolve inserting fragments of genomic DNA into an expression vector,such as a plasmid, transforming α-amylase-negative bacteria with theresulting genomic DNA library, and then plating the transformed bacteriaonto agar containing a substrate for α-amylase, thereby allowing clonesexpressing the α-amylase to be identified.

Alternatively, the DNA sequence encoding the enzyme may be preparedsynthetically by established standard methods, e.g., thephosphoroamidite method described by S. L. Beaucage and M. H. Caruthers(1981) or the method described by Matthes et al. (1984). In thephosphoroamidite method, oligonucleotides are synthesized, e.g., in anautomatic DNA synthesizer, purified, annealed, ligated and cloned inappropriate vectors.

Finally, the DNA sequence may be of mixed genomic and synthetic origin,mixed synthetic and cDNA origin or mixed genomic and cDNA origin,prepared by ligating fragments of synthetic, genomic or cDNA origin (asappropriate, the fragments corresponding to various parts of the entireDNA sequence), in accordance with standard techniques. The DNA sequencemay also be prepared by polymerase chain reaction (PCR) using specificprimers, for instance as described in U.S. Pat. No. 4,683,202 or R. K.Saiki et al. (1988).

Expression of α-Amylase Variants

According to the invention, a DNA sequence encoding the variant producedby methods described above, or by any alternative methods known in theart, can be expressed, in enzyme form, using an expression vector whichtypically includes control sequences encoding a promoter, operator,ribosome binding site, translation initiation signal, and, optionally, arepressor gene or various activator genes.

The recombinant expression vector carrying the DNA sequence encoding anα-amylase variant of the invention may be any vector which mayconveniently be subjected to recombinant DNA procedures, and the choiceof vector will often depend on the host cell into which it is to beintroduced. Thus, the vector may be an autonomously replicating vector,i.e., a vector which exists as an extrachromosomal entity, thereplication of which is independent of chromosomal replication, e.g., aplasmid, a bacteriophage or an extrachromosomal element, minichromosomeor an artificial chromosome. Alternatively, the vector may be one which,when introduced into a host cell, is integrated into the host cellgenome and replicated together with the chromosome(s) into which it hasbeen integrated.

In the vector, the DNA sequence should be operably connected to asuitable promoter sequence. The promoter may be any DNA sequence whichshows transcriptional activity in the host cell of choice and may bederived from genes encoding proteins either homologous or heterologousto the host cell. Examples of suitable promoters for directing thetranscription of the DNA sequence encoding an α-amylase variant of theinvention, especially in a bacterial host, are the promoter of the lacoperon of E. coli, the Streptomyces coelicolor agarase gene dagApromoters, the promoters of the Bacillus licheniformis α-amylase gene(amyL), the promoters of the Bacillus stearothermophilus maltogenicamylase gene (amyM), the promoters of the Bacillus amyloliquefaciensα-amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylBgenes etc. For transcription in a fungal host, examples of usefulpromoters are those derived from the gene encoding A. oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, A. niger neutralα-amylase, A. niger acid stable α-amylase, A. niger glucoamylase,Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triosephosphate isomerase or A. nidulans acetamidase.

The expression vector of the invention may also comprise a suitabletranscription terminator and, in eukaryotes, polyadenylation sequencesoperably connected to the DNA sequence encoding the α-amylase variant ofthe invention. Termination and polyadenylation sequences may suitably bederived from the same sources as the promoter.

The vector may further comprise a DNA sequence enabling the vector toreplicate in the host cell in question. Examples of such sequences arethe origins of replication of plasmids pUC19, pACYC177, pUB110, pE194,pAMB1 and pIJ702.

The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell, such as the dalgenes from B. subtilis or B. licheniformis, or one which confersantibiotic resistance such as ampicillin, kanamycin, chloramphenicol ortetracyclin resistance. Furthermore, the vector may comprise Aspergillusselection markers such as amdS, argB, niaD and sC, a marker giving riseto hygromycin resistance, or the selection may be accomplished byco-transformation, e.g., as described in WO 91/17243.

While intracellular expression may be advantageous in some respects,e.g., when using certain bacteria as host cells, it is generallypreferred that the expression is extracellular. In general, the Bacillusα-amylases mentioned herein comprise a pre-region permitting secretionof the expressed protease into the culture medium. If desirable, thispreregion may be replaced by a different preregion or signal sequence,conveniently accomplished by substitution of the DNA sequences encodingthe respective preregions.

The procedures used to ligate the DNA construct of the inventionencoding an α-amylase variant, the promoter, terminator and otherelements, respectively, and to insert them into suitable vectorscontaining the information necessary for replication, are well known topersons skilled in the art (cf., for instance, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor,1989).

The cell of the invention, either comprising a DNA construct or anexpression vector of the invention as defined above, is advantageouslyused as a host cell in the recombinant production of an α-amylasevariant of the invention. The cell may be transformed with the DNAconstruct of the invention encoding the variant, conveniently byintegrating the DNA construct (in one or more copies) in the hostchromosome. This integration is generally considered to be an advantageas the DNA sequence is more likely to be stably maintained in the cell.Integration of the DNA constructs into the host chromosome may beperformed according to conventional methods, e.g., by homologous orheterologous recombination. Alternatively, the cell may be transformedwith an expression vector as described above in connection with thedifferent types of host cells.

The cell of the invention may be a cell of a higher organism such as amammal or an insect, but is preferably a microbial cell, e.g. abacterial or a fungal (including yeast) cell.

Examples of suitable bacteria are Gram positive bacteria such asBacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillusbrevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacilluslautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyceslividans or Streptomyces murinus, or gramnegative bacteria such as E.coli. The transformation of the bacteria may, for instance, be effectedby protoplast transformation or by using competent cells in a mannerknown per se.

The yeast organism may favorably be selected from a species ofSaccharomyces or Schizosaccharomyces, e.g. Saccharomyces cerevisiae. Thefilamentous fungus may advantageously belong to a species ofAspergillus, e.g., Aspergillus oryzae or Aspergillus niger. Fungal cellsmay be transformed by a process involving protoplast formation andtransformation of the protoplasts followed by regeneration of the cellwall in a manner known per se. A suitable procedure for transformationof Aspergillus host cells is described in EP 238 023.

In a yet further aspect, the present invention relates to a method ofproducing an α-amylase variant of the invention, which method comprisescultivating a host cell as described above under conditions conducive tothe production of the variant and recovering the variant from the cellsand/or culture medium.

The medium used to cultivate the cells may be any conventional mediumsuitable for growing the host cell in question and obtaining expressionof the α-amylase variant of the invention. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedrecipes (e.g. as described in catalogues of the American Type CultureCollection).

The α-amylase variant secreted from the host cells may conveniently berecovered from the culture medium by well-known procedures, includingseparating the cells from the medium by centrifugation or filtration,and precipitating proteinaceous components of the medium by means of asalt such as ammonium sulphate, followed by the use of chromatographicprocedures such as ion exchange chromatography, affinity chromatography,or the like.

Industrial Applications

The α-amylase variants of this invention possesses valuable propertiesallowing for a variety of industrial applications. In particular, enzymevariants of the invention are applicable as a component in washing,dishwashing and hard-surface cleaning detergent compositions.

Numerous variants are particularly useful in the production ofsweeteners and ethanol from starch, and/or for textile desizing.Conditions for conventional starch-conversion processes, includingstarch liquefaction and/or saccharification processes, are described in,e.g., U.S. Pat. No. 3,912,590 and in EP patent publications Nos. 252,730and 63,909.

Detergent Compositions

As mentioned above, variants of the invention may suitably beincorporated in detergent compositions. Reference is made, for example,to WO 96/23874 and WO 97/07202 for further details concerning relevantingredients of detergent compositions (such as laundry or dishwashingdetergents), appropriate methods of formulating the variants in suchdetergent compositions, and for examples of relevant types of detergentcompositions.

Detergent compositions comprising a variant of the invention mayadditionally comprise one or more other enzymes, such as a lipase,cutinase, protease, cellulase, peroxidase or laccase, and/or anotherα-amylase.

α-amylase variants of the invention may be incorporated in detergents atconventionally employed concentrations. It is at present contemplatedthat a variant of the invention may be incorporated in an amountcorresponding to 0.00001-1 mg (calculated as pure, active enzymeprotein) of α-amylase per liter of wash/dishwash liquor usingconventional dosing levels of detergent.

The invention also relates to a method of providing α-amylases with 1)altered pH optimum, and/or 2) altered temperature optimum, and/or 3)improved stability, comprising the following steps:

-   i) identifying (a) target position(s) and/or region(s) for mutation    of the α-amylase by comparing the molecular dynamics of two or more    α-amylase 3D structures having substantially different pH,    temperature and/or stability profiles,-   ii) substituting, adding and/or deleting one or more amino acids in    the identified position(s) and/or region(s).

In embodiment of the invention a medium temperature α-amylase iscompared with a high temperature α-amylase. In another embodiment a lowtemperature α-amylase is compared with either a medium or a hightemperature α-amylase.

The α-amylases compared should preferably be at least 70%, preferably80%, up to 90%, such as up to 95%, especially 95% homologous with eachother.

The α-amylases compared may be Termamyl-like α-amylases as definedabove. In specific embodiment the α-amylases compared are the α-amylasesshown in SEQ ID NO: 1 to SEQ ID NO: 8.

In another embodiment the stability profile of the α-amylases inquestion compared are the Ca²⁺ dependency profile.

Materials and Methods

Enzymes:

-   SP722: (SEQ ID NO: 2, available from Novo Nordisk)-   Termamyl™ (SEQ ID NO: 4, available from Novo Nordisk)-   SP690: (SEQ ID NO: 1, available from Novo Nordisk)-   Bacillus subtilis SHA273: see WO 95/10603    Plasmids

pJE1 contains the gene encoding a variant of SP722 α-amylase (SEQ ID NO:2): viz. deletion of 6 nucleotides corresponding to amino acidsD183-G184 in the mature protein. Transcription of the JE1 gene isdirected from the amyL promoter. The plasmid further more contains theorigin of replication and cat-gene conferring resistance towardskanamycin obtained from plasmid pUB110 (Gryczan, T J et al. (1978), J.Bact. 134:318-329).

Methods:

Construction of Library Vector pDorK101

The E. coli/Bacillus shuttle vector pDorK101 (described below) can beused to introduce mutations without expression of α-amylase in E. coliand then be modified in such way that the α-amylase is active inBacillus. The vector was constructed as follows: The JE1 encoding gene(SP722 with the deletion of D183-G184) was inactivated in pJE1 by geneinterruption in the PstI site in the 5′ coding region of the SEQ ID NO:2: SP722 by a 1.2 kb fragment containing an E. coli origin ofreplication. This fragment was PCR amplified from the pUC19 (GenBankAccession #:X02514) using the forward primer: 5′-gacctgcagtcaggcaacta-3′and the reverse primer: 5′-tagagtcgacctgcaggcat-3′. The PCR amplicon andthe pJE1 vector were digested with PstI at 37° C. for 2 hours. The pJE1vector fragment and the PCR fragment were ligated at room temperature.for 1 hour and transformed in E. coli by electrotransformation. Theresulting vector is designated pDorK101.

Filter Screening Assays

The assay can be used to screening of Termamyl-like α-amylase variantshaving an improved stability at high pH compared to the parent enzymeand Termamyl-like α-amylase variants having an improved stability athigh pH and medium temperatures compared to the parent enzyme dependingof the screening temperature setting.

High pH Filter Assay

Bacillus libraries are plated on a sandwich of cellulose acetate (OE 67,Schleicher & Schuell, Dassel, Germany)—and nitrocellulose filters(Protran-Ba 85, Schleicher & Schuell, Dassel, Germany) on TY agar plateswith 10 μg/ml kanamycin at 37° C. for at least 21 hours. The celluloseacetate layer is located on the TY agar plate.

Each filter sandwich is specifically marked with a needle after plating,but before incubation in order to be able to localize positive variantson the filter and the nitrocellulose filter with bound variants istransferred to a container with glycin-NaOH buffer, pH 8.6-10.6 andincubated at room temperature (can be altered from 10′-60° C.) for 15min. The cellulose acetate filters with colonies are stored on theTY-plates at room temperature until use. After incubation, residualactivity is detected on plates containing 1% agarose, 0.2% starch inglycin-NaOH buffer, pH 8.6-10.6. The assay plates with nitrocellulosefilters are marked the same way as the filter sandwich and incubated for2 hours. at room temperature. After removal of the filters the assayplates are stained with 10% Lugol solution. Starch degrading variantsare detected as white spots on dark blue background and then identifiedon the storage plates. Positive variants are rescreened twice under thesame conditions as the first screen.

Low Calcium Filter Assay

The Bacillus library are plated on a sandwich of cellulose acetate (OE67, Schleicher & Schuell, Dassel, Germany)—and nitrocellulose filters(Protran-Ba 85, Schleicher & Schuell, Dassel, Germany) on TY agar plateswith a relevant antibiotic, e.g., kanamycin or chloramphenicol, at 37°C. for at least 21 hours. The cellulose acetate layer is located on theTY agar plate.

Each filter sandwich is specifically marked with a needle after plating,but before incubation in order to be able to localize positive variantson the filter and the nitrocellulose filter with bound variants istransferred to a container with carbonate/bicarbonate buffer pH 8.5-10and with different EDTA concentrations (0.001 mM-100 mM). The filtersare incubated at room temperature for 1 hour. The cellulose acetatefilters with colonies are stored on the TY-plates at room temperatureuntil use. After incubation, residual activity is detected on platescontaining 1% agarose, 0.2% starch in carbonate/bicarbonate buffer pH8.5-10. The assay plates with nitrocellulose filters are marked the sameway as the filter sandwich and incubated for 2 hours. at roomtemperature. After removal of the filters the assay plates are stainedwith 10% Lugol solution. Starch degrading variants are detected as whitespots on dark blue background and then identified on the storage plates.Positive variants are rescreened twice under the same conditions as thefirst screen.

Method to Obtaining the Regions of Interest:

There are three known 3D structures of bacterial α-amylases. Two of B.licheniformis α-amylase, Brookhaven database 1BPL (Machius et al.(1995), J. Mol. Biol. 246, p. 545-559) and 1VJS (Song et al. (1996),Enzymes for Carbohydrate 163 Engineering (Prog. Biotechnol. V 12). Thesetwo structures are lacking an important piece of the structure from theso-called B-domain, in the area around the two Calcium ions and oneSodium ion binding sites. We have therefore used a 3D structure of anα-amylase BA2 (WO 96/23874 which are a hybrid between BAN™ (SEQ ID NO.5) and B. licheniformis α-amylase (SEQ ID NO. 4). On basis of thestructure a model of B. licheniformis alpha amylase and the SP722α-amylase has been build.

Fermentation and Purification of α-Amylase Variants

Fermentation and purification may be performed by methods well known inthe art.

Stability Determination

All stability trials are made using the same set up. The method are:

The enzyme is incubated under the relevant conditions (1-4). Samples aretaken at various time points, e.g., after 0, 5, 10, 15 and 30 minutesand diluted 25 times (same dilution for all taken samples) in assaybuffer (0.1M 50 mM Britton buffer pH 7.3) and the activity is measuredusing the Phadebas assay (Pharmacia) under standard conditions pH 7.3,37° C.

The activity measured before incubation (0 minutes) is used as reference(100%). The decline in percent is calculated as a function of theincubation time. The table shows the residual activity after, e.g., 30minutes of incubation.

Specific Activity Determination

The specific activity is determined using the Phadebas assay (Pharmacia)as activity/mg enzyme. The manufactures instructions are followed (seealso below under “Assay for α-amylase activity).

Assays for α-Amylase Activity

1. Phadebas Assay

α-amylase activity is determined by a method employing Phadebas® tabletsas substrate. Phadebas tablets (Phadebas® Amylase Test, supplied byPharmacia Diagnostic) contain a cross-linked insoluble blue-coloredstarch polymer which has been mixed with bovine serum albumin and abuffer substance and tabletted.

For every single measurement one tablet is suspended in a tubecontaining 5 ml 50 mM Britton-Robinson buffer (50 mM acetic acid, 50 mMphosphoric acid, 50 mM boric acid, 0.1 mM CaCl₂, pH adjusted to thevalue of interest with NaOH). The test is performed in a water bath atthe temperature of interest. The α-amylase to be tested is diluted in xml of 50 mM Britton-Robinson buffer. 1 ml of this α-amylase solution isadded to the 5 ml 50 mM Britton-Robinson buffer. The starch ishydrolyzed by the α-amylase giving soluble blue fragments. Theabsorbance of the resulting blue solution, measuredspectrophotometrically at 620 nm, is a function of the α-amylaseactivity.

It is important that the measured 620 nm absorbance after 10 or 15minutes of incubation (testing time) is in the range of 0.2 to 2.0absorbance units at 620 nm. In this absorbance range there is linearitybetween activity and absorbance (Lambert-Beer law). The dilution of theenzyme must therefore be adjusted to fit this criterion. Under aspecified set of conditions (temp., pH, reaction time, bufferconditions) 1 mg of a given α-amylase will hydrolyze a certain amount ofsubstrate and a blue colour will be produced. The colour intensity ismeasured at 620 nm. The measured absorbance is directly proportional tothe specific activity (activity/mg of pure α-amylase protein) of theα-amylase in question under the given set of conditions.

2. Alternative Method

α-amylase activity is determined by a method employing the PNP-G7substrate. PNP-G7 which is a abbreviation forp-nitrophenyl-α,D-maltoheptaoside is a blocked oligosaccharide which canbe cleaved by an endo-amylase. Following the cleavage, the α-Glucosidaseincluded in the kit digest the substrate to liberate a free PNP moleculewhich has a yellow colour and thus can be measured by visiblespectophometry at λ=405 nm. (400-420 nm.). Kits containing PNP-G7substrate and α-Glucosidase is manufactured by Boehringer-Mannheim (cat.No. 1054635).

To prepare the substrate one bottle of substrate (BM 1442309) is addedto 5 ml buffer (BM1442309). To prepare the α-Glucosidase one bottle ofα-Glucosidase (BM 1462309) is added to 45 ml buffer (BM1442309). Theworking solution is made by mixing 5 ml α-Glucosidase solution with 0.5ml substrate.

The assay is performed by transforming 201 enzyme solution to a 96 wellmicrotitre plate and incubating at 25° C. 200 μl working solution, 25°C. is added. The solution is mixed and pre-incubated 1 minute andabsorption is measured every 15 sec. over 3 minutes at OD 405 nm.

The slope of the time dependent absorption-curve is directlyproportional to the specific activity (activity per mg enzyme) of theα-amylase in question under the given set of conditions.

General Method for Random Mutagenesis by Use of the DOPE Program

The random mutagenesis may be carried out by the following steps:

-   1. Select regions of interest for modification in the parent enzyme-   2. Decide on mutation sites and non-mutated sites in the selected    region-   3. Decide on which kind of mutations should be carried out, e.g.    with respect to the desired stability and/or performance of the    variant to be constructed-   4. Select structurally reasonable mutations.-   5. Adjust the residues selected by step 3 with regard to step 4.-   6. Analyze by use of a suitable dope algorithm the nucleotide    distribution.-   7. If necessary, adjust the wanted residues to genetic code realism    (e.g., taking into account constraints resulting from the genetic    code (e.g. in order to avoid introduction of stop codons))(the    skilled person will be aware that some codon combinations cannot be    used in practice and will need to be adapted)-   8. Make primers-   9. Perform random mutagenesis by use of the primers-   10. Select resulting α-amylase variants by screening for the desired    improved properties.

Suitable dope algorithms for use in step 6 are well known in the art.One algorithm is described by Tomandl, D. et al., Journal ofComputer-Aided Molecular Design, 11 (1997), pp. 29-38). Anotheralgorithm, DOPE, is described in the following:

The Dope Program

The “DOPE” program is a computer algorithm useful to optimize thenucleotide composition of a codon triplet in such a way that it encodesan amino acid distribution which resembles most the wanted amino aciddistribution. In order to assess which of the possible distributions isthe most similar to the wanted amino acid distribution, a scoringfunction is needed. In the “Dope” program the following function wasfound to be suited:${s \equiv {\prod\limits_{i = 1}^{N}\left( {\frac{x_{i}^{y_{i}}}{y_{i}^{y_{i}}}\frac{\left( {1 - x_{i}} \right)^{1 - y_{i}}}{\left( {1 - y_{i}} \right)^{1 - y_{i}}}} \right)^{w_{i}}}},$where the x_(i)'s are the obtained amounts of amino acids and groups ofamino acids as calculated by the program, y_(i)'s are the wanted amountsof amino acids and groups of amino acids as defined by the user of theprogram (e.g. specify which of the 20 amino acids or stop codons arewanted to be introduced, e.g. with a certain percentage (e.g. 90% Ala,3% Ile, 7% Val), and w_(i)'s are assigned weight factors as defined bythe user of the program (e.g., depending on the importance of having aspecific amino acid residue inserted into the position in question). Nis 21 plus the number of amino acid groups as defined by the user of theprogram. For purposes of this function 0⁰ is defined as being 1.

A Monte-Carlo algorithm (one example being the one described by Valleau,J. P. & Whittington, S. G. (1977) A guide to Mont Carlo for statisticalmechanics: 1 Highways. In “Stastistical Mechanics, Part A” EqulibriumTechniqeues ed. B. J. Berne, New York: Plenum) is used for finding themaximum value of this function. In each iteration the following stepsare performed:

-   1. A new random nucleotide composition is chosen for each base,    where the absolute difference between the current and the new    composition is smaller than or equal to d for each of the four    nucleotides G,A,T,C in all three positions of the codon (see below    for definition of d).-   2. The scores of the new composition and the current composition are    compared by the use of the function s as described above. If the new    score is higher or equal to the score of the current composition,    the new composition is kept and the current composition is changed    to the new one. If the new score is smaller, the probability of    keeping the new composition is    exp(1000(new_score−current_score)).

A cycle normally consists of 1000 iterations as described above in whichd is decreasing linearly from 1 to 0. One hundred or more cycles areperformed in an optimization process. The nucleotide compositionresulting in the highest score is finally presented.

EXAMPLES Example 1 Example on Homology Building of Termamyl™

The overall homology of the B. licheniformis α-amylase (in the followingreferred to as Termamyl™) to other Termamyl-like α-amylases is high andthe percent similarity is extremely high. The similarity calculated forTermamyl™ to BSG (the B. stearothermophilus α-amylase having SEQ ID NO:3), and BAN (the B. amyloliquefaciens α-amylase having SEQ ID NO: 5)using the University of Wisconsin Genetics Computer Group's program GCGgave 89% and 78%, respectively. TERM has a deletion of 2 residuesbetween residue G180 and K181 compared to BAN™ and BSG. BSG has adeletion of 3 residues between G371 and 1372 in comparison with BAN™ andTermamyl™. Further BSG has a C-terminal extension of more than 20residues compared to BAN™ and Termamyl™. BAN™ has 2 residues less andTermamyl has one residue less in the N-terminal compared to BSG.

The structure of the B. licheniformis (Termamyl™) and of the B.amyloliquefaciens α-amylase (BAN™), respectively, was model built on thestructure disclosed in Appendix 1 of WO 96/23974. The structure of otherTermamyl-like α-amylases (e.g. those disclosed herein) may be builtanalogously.

In comparison with the α-amylase used for elucidating the presentstructure, Termamyl™ differs in that it lacks two residues around178-182. In order to compensate for this in the model structure, theHOMOLOGY program from BIOSYM was used to substitute the residues inequivalent positions in the structure (not only structurally conservedregions) except for the deletion point. A peptide bond was establishedbetween G179(G177) and K180(K180) in Termamyl™(BAN™). The closestructural relationship between the solved structure and the modelstructure (and thus the validity of the latter) is indicated by thepresence of only very few atoms found to be too close together in themodel.

To this very rough structure of Termamyl™ was then added all waters(605) and ions (4 Calcium and 1 Sodium) from the solved structure (SeeAppendix 1 of WO 96/23874) at the same coordinates as for said solvedstructure using the INSIGHT program. This could be done with only fewoverlaps—in other words with a very nice fit. This model structure werethen minimized using 200 steps of Steepest descent and 600 steps ofConjugated gradient (see Brooks et al 1983, J. Computational Chemistry4, p. 187-217). The minimized structure was then subjected to moleculardynamics, 5 ps heating followed by up to 200 ps equilibration but morethan 35 ps. The dynamics as run with the Verlet algorithm and theequilibration temperature 300K were kept using the Behrendsen couplingto a water bath (Berendsen et. al., 1984, J. Chemical Physics 81, p.3684-3690). Rotations and translations were removed every pico second.

Example 2 Method of Extracting Important Regions for Identifyingα-Amylase Variants with Improved pH Stability and Altered TemperatureActivity

The X-ray structure and/or the model build structure of the enzyme ofinterest, here SP722 and Termamyl™, are subjected to molecular dynamicssimulations. The molecular dynamics simulation are made using the CHARMM(from Molecular simulations (MSI)) program or other suited program like,e.g., DISCOVER (from MSI). The molecular dynamic analysis is made invacuum, or more preferred including crystal waters, or with the enzymeembedded in water, e.g., a water sphere or a water box. The simulationare run for 300 pico seconds (ps) or more, e.g., 300-1200 ps. Theisotropic fluctuations are extracted for the CA carbons of thestructures and compared between the structures. Where the sequence hasdeletions and/or insertions the isotropic fluctuations from the otherstructure are inserted thus giving 0 as difference in isotropicfluctuation. For explanation of isotropic fluctuations see the CHARMMmanual (obtainable from MSI).

The molecular dynamics simulation can be done using standard charges onthe chargeable amino acids. This is Asp and Glu are negatively chargedand Lys and Arg are positively charged. This condition resembles themedium pH of approximately 7. To analyze a higher or lower pH, titrationof the molecule can be done to obtain the altered pKa's of the standardtitrateable residues normally within pH 2-10; Lys, Arg, Asp, Glu, Tyrand His. Also Ser, Thr and Cys are titrateable but are not taking intoaccount here. Here the altered charges due to the pH has been describedas both Asp and Glu are negative at high pH, and both Arg and Lys areuncharged. This imitates a pH around 10 to 11 where the titration of Lysand Arg starts, as the normal pKa of these residues are around 9-11.

1. The approach used for extracting important regions for identifyingα-amylase variants with high pH stability:

The important regions for constructing variants with improved pHstability are the regions which at the extreme pH display the highestmobility, i.e., regions having the highest isotropic fluctuations.

Such regions are identified by carrying out two molecular dynamicssimulations: i) a high pH run at which the basic amino acids, Lys andArg, are seen as neutral (i.e. not protonated) and the acidic aminoacids, Asp and Glu, have the charge (−1) and ii) a neutral pH run withthe basic amino acids, Lys and Arg, having the net charge of (+1) andthe acidic amino acids having a charge of (−1).

The two run are compared and regions displaying the relatively highermobility at high pH compared to neutral pH analysis were identified.

Introduction of residues improving general stability, e.g., hydrogenbonding, making the region more rigid (by mutations such as Prolinesubstitutions or replacement of Glycine residues), or improving thecharges or their interaction, improves the high pH stability of theenzyme.

2. The approach used for extracting regions for identifying α-amylasevariants with increased activity at medium temperatures:

The important regions for constructing variants with increased activityat medium temperature was found as the difference between the isotropicfluctuations in SP722 and Termamyl, i.e., SP722 minus Termamyl CAisotrophic fluctuations, The regions with the highest mobility in theisotrophic fluctuations were selected. These regions and there residueswere expected to increase the activity at medium temperatures. Theactivity of an alpha-amylase is only expressed if the correct mobilityof certain residues are present. If the mobility of the residues is toolow the activity is decreased or abandoned.

Example 3 Construction, by Localized Random, Doped Mutagenesis, ofTermamyl-Like α-Amylase Variants Having an Improved Ca2+ Stability atMedium Temperatures Compared to the Parent Enzyme

To improve the stability at low calcium concentration of α-amylasesrandom mutagenesis in pre-selected region was performed.

-   Region: Residue:-   SAI: R181-W189

The DOPE software (see Materials and Methods) was used to determinespiked codons for each suggested change in the SA1 region minimizing theamount of stop codons (see table 1). The exact distribution ofnucleotides was calculated in the three positions of the codon to givethe suggested population of amino acid changes. The doped regions weredoped specifically in the indicated positions to have a high chance ofgetting the desired residues, but still allow other possibilities. TABLE1 Distribution of amino acid residues for each position R181:  72% R, 2%N, 7% Q, 4% H, 4% K, 11% S G182:  73% G, 13% A, 12% S, 2% T K185:  95%K, 5% R A186:  50% A, 4% N, 6% D, 1% E, 1% G, 1% K, 5% S, 31% T W187:100% W D188: 100% D W189:  92% W, 8% S

The resulting doped oligonucleotide strand is shown in table 2 as sensestrand: with the wild type nucleotide and amino acid sequences and thedistribution of nucleotides for each doped position. TABLE 2 Position181 182 185 186 187 188 189 Amino acid seq. Arg Gly Lys Ala Thr Asp ThrWt nuc. seq. cga ggt aaa gct tgg gat tgg Forward primer: FSA: (SEQ IDNO: 15) 5′-caa aat cgt atc tac aaa ttc 123 456 a7g 8910 tgg gat t11g gaagta gat tcg gaa aat-3′

Distribution of Nucleotides for each Doped Position 1: 35% A, 65% C 2:83% G, 17% A 3: 63% G, 37% T 4: 86% G, 14% A 5: 85% G, 15% C 6: 50% T,50% C 7: 95% A, 5% G 8: 58% G, 37% A, 5% T 9: 86% C, 13% A, 1% G 10: 83%T, 17% G 11: 92% G, 8% C

Reverse primer: RSA: 5′-gaa ttt gta gat acg att ttg-3′ (SEQ ID NO: 16)Random Mutagenesis

The spiked oligonucleotides apparent from Table 2 (which by a commonterm is designated FSA) and reverse primers RSA for the SA1 region andspecific SEQ ID NO: 2: SP722 primers covering the SacII and the DraIIIsites are used to generate PCR-library-fragments by the overlapextension method (Horton et al., Gene, 77 (1989), pp. 61-68) with anoverlap of 21 base pairs. Plasmid pJE1 is template for the PolymeraseChain Reaction. The PCR fragments are cloned in the E. coli/Bacillusshuttle vector pDork101 (see Materials and Methods) enabling mutagenesisin E. coli and immediate expression in Bacillus subtilis preventinglethal accumulation of amylases in E. coli. After establishing thecloned PCR fragments in E. coli, a modified pUC19 fragment is digestedout of the plasmid and the promoter and the mutated Termamyl gene isphysically connected and expression can take place in Bacillus.

Screening

The library may be screened in the low calcium filter assays describedin the “Material and Methods” section above.

Example 4 Construction of Variants of Amylase SEQ ID NO: 1 (SP690)

The gene encoding the amylase from SEQ ID NO: 1 is located in a plasmidpTVB106 described in WO96/23873. The amylase is expressed from the amyLpromoter in this construct in Bacillus subtilis.

A variant of the protein is delta(T183−G184)+Y243F+Q391E+K444Q.Construction of this variant is described in WO96/23873.

Construction of delta(T183−G184)+N195F by the mega-primer method asdescribed by Sarkar and Sommer, (1990), BioTechniques 8: 404-407.

Gene specific primer B1 (SEQ ID NO: 17) and mutagenic primer 101458 (SEQID NO: 19) were used to amplify by PCR an approximately 645 bp DNAfragment from a pTVB106-like plasmid (with the delta(T183−G184)mutations in the gene encoding the amylase from SEQ ID NO: 1).

The 645 bp fragment was purified from an agarose gel and used as amega-primer together with primer Y2 (SEQ ID NO: 18) in a second PCRcarried out on the same template.

The resulting approximately 1080 bp fragment was digested withrestriction enzymes BstEII and AflIII and the resulting approximately510 bp DNA fragment was purified and ligated with the pTVB106-likeplasmid (with the delta(T183−G184) mutations in the gene encoding theamylase from SEQ ID NO: 1) digested with the same enzymes. CompetentBacillus subtilis SHA273 (amylase and protease low) cells weretransformed with the ligation and Chlorampenicol resistant transformantsand was checked by DNA sequencing to verify the presence of the correctmutations on the plasmid. primer B1: 5′ CGA TTG CTG ACG CTG TTA (SEQ IDNO: 17) TTT GCG 3′ primer Y2: 5′ CTT GTT CCC TTG TCA GAA (SEQ ID NO: 18)CCA ATG 3′ primer 101458: 5′ GT CAT AGT TGC CGA AAT CTG (SEQ ID NO: 19)TAT CGA CTT C 3′

The construction of variant: delta(T183−G184)+K185R+A186T was carriedout in a similar way except that mutagenic primer 101638 was used.primer 101638: 5′ CC CAG TCC CAC GTA CGT CCC (SEQ ID NO: 20) CTG AAT TTATAT ATT TTG 3′

Variants: delta(T183−G184)+A186T, delta(T183−G184)+A186I,delta(T183−G184)+A186S, delta(T183−G184)+A186N are constructed by asimilar method except that pTVB106-like plasmid (carrying variantdelta(T183−G184)+K185R+A186T) is used as template and as the vector forthe cloning purpose. The mutagenic oligonucleotide (Oligo 1) is: 5′ CCCAG TCC CAG NTCTTT CCC (SEQ ID NO: 21) CTG AAT TTA TAT ATT TTG 3′

N represents a mixture of the four bases: A, C, G, and T used in thesynthesis of the mutagenicoli-gonucleotide. Sequencing of transformantsidentifies the correct codon for amino acid position 186 in the matureamylase.

Variant: delta(T183−G184)+K185R+A186T+N195F is constructed as follows:

PCR is carried out with primer x2 (SEQ ID NO: 22) and primer 101458 (SEQID NO: 19) on pTVB106-like plasmid (with mutationsdelta(T183−G184)+K185R+A186T). The resulting DNA fragment is used as amega-primer together with primer Y2 (SEQ ID NO: 18) in a PCR onpTVB106-like plasmid (with mutations delta(T183−G184)+N195). The productof the second PCR is digested with restriction endonucleases Acc65I andAflIII and cloned into pTVB106 like plasmid (delta(T183−G184)+N195F)digested with the same enzymes.

primer x2: (SEQ ID NO: 22)

5′ GCG TGG ACA AAG TTT GAT TTT CCT G 3′

Variant: delta(T183−G184)+K185R+A186T+N195F+Y243F+Q391E+K444Q isconstructed as follows:

PCR is carried out with primer x2 and primer 101458 on pTVB106-likeplasmid (with mutations delta(T183−G184)+K185R+A186T). The resulting DNAfragment is used as a mega-primer together with primer Y2 in a PCR onpTVB106 like plasmid (with mutationsdelta(T183−G184)+Y243F+Q391E+K444Q). The product of the second PCR isdigested with restriction endonucleases Acc65I and AflIII and clonedinto pTVB106 like plasmid (delta(T183−G184)+Y243F+Q391E+K444Q) digestedwith the same enzymes.

Example 5 Construction of Site-Directed α-Amylase Variants in the ParentSP722 α-Amylase (SEQ ID NO: 2)

Construction of variants of amylase SEQ ID NO: 2 (SP722) is carried outas described below.

The gene encoding the amylase from SEQ ID NO: 2 is located in a plasmidpTVB112 described in WO 96/23873. The amylase is expressed from the amyLpromoter in this construct in Bacillus subtilis.

Construction of delta(D183−G184)+V56I by the mega-primer method asdescribed by Sarkar and Sommer, 1990 (BioTechniques 8: 404-407).

Gene specific primer DA03 and mutagenic primer DA07 are used to amplifyby PCR an approximately 820 bp DNA fragment from a pTVB112-like plasmid(with the delta(D183−G184) mutations in the gene encoding the α-amylaseshown in SEQ ID NO: 2.

The 820 bp fragment is purified from an agarose gel and used as amega-primer together with primer DA01 in a second PCR carried out on thesame template.

The resulting approximately 920 bp fragment is digested with restrictionenzymes NgoM I and Aat II and the resulting approximately 170 bp DNAfragment is purified and ligated with the pTVB112-like plasmid (with thedelta(D183−G184) mutations in the gene encoding the amylase shown in SEQID NO: 2) digested with the same enzymes. Competent Bacillus subtilisSHA273 (amylase and protease low) cells are transformed with theligation and Chlorampenicol resistant transformants are checked by DNAsequencing to verify the presence of the correct mutations on theplasmid. primer DA01: 5′ CCTAATGATGGGAATCACTGG 3′ (SEQ ID NO:23) primerDA03: 5′ GCATTGGATGCTTTTGAACAACCG 3′ (SEQ ID NO:24) primer DA07: 5′CGCAAAATGATATCGGGTATGGAGCC 3′ (SEQ ID NO:25)Variants: delta(D183−G184)+K108L, delta(D183−G184)+K108Q,delta(D183−G184)+K108E, delta(D183−G184)+K108V, were constructed by themega-primer method as described by Sarkar and Sommer, 1990(BioTechniques 8: 404-407):

PCR is carried out with primer DA03 and mutagenesis primer DA20 onpTVB112-like plasmid (with mutations delta(D183−G184)). The resultingDNA fragment is used as a mega-primer together with primer DA01 in a PCRon pTVB112-like plasmid (with mutations delta(D183−G184)). Theapproximately 920 bp product of the second PCR is digested withrestriction endonucleases Aat II and Mlu I and cloned into pTVB112-likeplasmid (delta(D183−G184)) digested with the same enzymes. primer DA20:5′ GTGATGAACCACSWAGGTGGAGCTGATGC 3′ (SEQ ID NO:26)

S represents a mixture of the two bases: C and G used in the synthesisof the mutagenic oligonucleotide and W represents a mixture of the twobases: A and T used in the synthesis of the mutagenic oligonucleotide.

Sequencing of transformants identifies the correct codon for amino acidposition 108 in the mature amylase.

Construction of the variants: delta(D183−G184)+D168A,delta(D183−G184)+D168I, delta(D183−G184)+D168V, delta(D183−G184)+D168Tis carried out in a similar way except that mutagenic primer DA14 isused. primer DA14: 5′ GATGGTGTATGGRYCAATCACGACAATTCC 3′ (SEQ ID NO:27)

R represents a mixture of the two bases: A and G used in the synthesisof the mutagenic oligonucleotide and Y represents a mixture of the twobases: C and T used in the synthesis of the mutagenic oligonucleotide.

Sequencing of transformants identifies the correct codon for amino acidposition 168 in the mature amylase.

Construction of the variant: delta(D183−G184)+Q169N is carried out in asimilar way except that mutagenic primer DA15 is used. primer DA15: 5′GGTGTATGGGATAACTCACGACAATTCC 3′ (SEQ ID NO:28)

Construction of the variant: delta(D183−G184)+Q169L is carried out in asimilar way except that mutagenic primer DA16 is used. primer DA16: 5′GGTGTATGGGATCTCTCACGACAATTCC 3′ (SEQ ID NO:29)

Construction of the variant: delta(D183−G184)+Q172N is carried out in asimilar way except that mutagenic primer DA17 is used. (SEQ ID NO:30)primer DA17: 5′ GGGATCAATCACGAAATTTCCAAAATCGTATC 3′

Construction of the variant: delta(D183−G184)+Q172L is carried out in asimilar way except that mutagenic primer DA18 is used. (SEQ ID NO:31)primer DA18: 5′ GGGATCAATCACGACTCTTCCAAAATCGTATC 3′

Construction of the variant: delta(D183−G184)+L201I is carried out in asimilar way except that mutagenic primer DA06 is used. (SEQ ID NO:32)primer DA06: 5′ GGAAATTATGATTATATCATGTATGCAGATGTAG 3′

Construction of the variant: delta(D183−G184)+K269S is carried out in asimilar way except that mutagenic primer DA09 is used. primer DA09: 5′GCTGAATTTTGGTCGAATGATTTAGGTGCC 3′ (SEQ ID NO:33)

Construction of the variant: delta(D183−G184)+K269Q is carried out in asimilar way except that mutagenic primer DA11 is used. primer DA11: 5′GCTGAATTTTGGTCGAATGATTTAGGTGCC 3′ (SEQ ID NO:34)

Construction of the variant: delta(D183−G184)+N270Y is carried out in asimilar way except that mutagenic primer DA21 is used. primer DA21: 5′GAATTTTGGAAGTACGATTTAGGTCGG 3′ (SEQ ID NO:35)

Construction of the variants: delta(D183−G184)+L272A,delta(D183−G184)+L272I, delta(D183−G184)+L272V, delta(D183−G184)+L272Tis carried out in a similar way except that mutagenic primer DA12 isused. primer DA12: 5′ GGAAAAACGATRYCGGTGCCTTGGAGAAC 3′ (SEQ ID NO:36)R represents a mixture of the two bases: A and G used in the synthesisof the mutagenic oligonucleotide and Y represents a mixture of the twobases: C and T used in the synthesis of the mutagenic oligonucleotide.Sequencing of transformants identifies the correct codon for amino acidposition 272 in the mature amylase.

Construction of the variants: delta(D183−G184)+L275A,delta(D183−G184)+L275I, delta(D183−G184)+L275V, delta(D183−G184)+L275Tis carried out in a similar way except that mutagenic primer DA13 isused. primer DA13: 5′ GATTTAGGTGCCTRYCAGAACTATTTA 3′ (SEQ ID NO:37)R represents a mixture of the two bases: A and G used in the synthesisof the mutagenic oligonucleotide and Y represents a mixture of the twobases: C and T used in the synthesis of the mutagenic oligonucleotide.Sequencing of transformants identifies the correct codon for amino acidposition 275 in the mature amylase.

Construction of the variant: delta(D183−G184)+Y295E is carried out in asimilar way except that mutagenic primer DA08 is used. primer DA08: 5′CCCCCTTCATGAGAATCTTTATAACG 3′ (SEQ ID NO:38)

Construction of delta(D183−G184)+K446Q by the mega-primer method asdescribed by Sarkar and Sommer, 1990 (BioTechniques 8: 404-407):

Gene specific primer DA04, annealing 214-231 bp downstream relative tothe STOP-codon and mutagenic primer DA10 were used to amplify by PCR anapproximately 350 bp DNA fragment from a pTVB112-like plasmid (with thedelta(D183−G184) mutations in the gene encoding the amylase depicted inSEQ ID NO: 2).

The resulting DNA fragment is used as a mega-primer together with primerDA05 in a PCR on pTVB112 like plasmid (with mutations delta(D183−G184)).The app. 460 bp product of the second PCR is digested with restrictionendonucleases SnaB I and Not I and cloned into pTVB112 like plasmid(delta(D183−G184)) digested with the same enzymes. primer DA04: 5′GAATCCGAACCTCATTACACATTCG 3′ (SEQ ID NO:39) primer DA05: 5′CGGATGGACTCGAGAAGGAAATACCACG 3′ (SEQ ID NO:40) primer DA10: 5′CGTAGGGCAAAATCAGGCCGGTCAAGTTT (SEQ ID NO:41) GG 3′

Construction of the variants: delta(D183−G184)+K458R is carried out in asimilar way except that mutagenic primer DA22 is used. primer DA22: 5′CATAACTGGAAATCGCCCGGGAACAGTTA (SEQ ID NO:42) CG 3′

Construction of the variants: delta(D183−G184)+P459S anddelta(D183−G184)+P459T is carried out in a similar way except thatmutagenic primer DA19 is used. primer DA19: 5′CTGGAAATAAAWCCGGAACAGTTACG 3′ (SEQ ID NO:43)W represents a mixture of the two bases: A and T used in the synthesisof the mutagenic oligonucleotide. Sequencing of transformants identifiesthe correct codon for amino acid position 459 in the mature amylase.

Construction of the variants: delta(D183−G184)+T461P is carried out in asimilar way except that mutagenic primer DA23 is used. primer DA23: 5′GGAAATAAACCAGGACCCGTTACGATCAAT (SEQ ID NO:44) GC 3′

Construction of the variant: delta(D183−G184)+K142R is carried out in asimilar way except that mutagenic primer DA32 is used. Primer DA32: 5′GAGGCTTGGACTAGGTTTGATTTTCCAG 3′ (SEQ ID NO:45)

Construction of the variant: delta(D183−G184)+K269R is carried out in asimilar way except that mutagenic primer DA31 is used. Primer DA31: 5′GCTGAATTTTGGCGCAATGATTTAGGTGCC 3′ (SEQ ID NO:46)

Example 6 Construction of Site-Directed α-Amylase Variants in the ParentTermamyl α-Amylase (SEQ ID NO: 4)

The amyL gene, encoding the Termamyl α-amylase is located in plasmidpDN1528 described in WO 95/10603 (Novo Nordisk). Variants withsubstitutions N265R and N265D, respectively, of said parent α-amylaseare constructed by methods described in WO 97/41213 or by the“megaprimer” approach described above.

Mutagenic Oligonucleotides are: (SEQ ID NO: 56) Primer bl1 for the N265Rsubstitution: 5′ PCC AGC GCG CCT AGG TCA CGC TGC CAA TAT TCA G (SEQ IDNO: 57) Primer bl2 for the N265D substitution: 5′ PCC AGC GCG CCT AGGTCA TCC TGC CAA TAT TCA GP represents a phosphate group.

Example 7 Determination of pH Stability at Alkaline pH of Variants ofthe Parent α-Amylase having the Amino Acid Sequence Shown in SEQ ID NO:2.

In this serie of analysis purified enzyme samples were used. Themeasurements were made using solutions of the respective variants in 100mM CAPS buffer adjusted to pH 10.5. The solutions were incubated at 75°C.

After incubation for 20 and 30 min the residual activity was measuredusing the PNP-G7 assay (described in the “Materials and Methods” sectionabove). The residual activity in the samples was measured using BrittonRobinson buffer pH 7.3. The decline in residual activity was measuredrelative to a corresponding reference solution of the same enzyme at 0minutes, which has not been incubated at high pH and 75° C.

The percentage of the initial activity as a function is shown in thetable below for the parent enzyme (SEQ ID NO: 2) and for the variants inquestion. Residual activity Residual activity Variant after 20 min after30 min Δ(D183-G184) + M323L 56% 44% Δ(D183-G184) + M323L + R181S 67% 55%Δ(D183-G184) + M323L + A186T 62% 50%

In an other series of analysis culture supernatants were used. Themeasurements were made using solutions of the respective variants in 100mM CAPS buffer adjusted to pH 10.5. The solutions were incubated at 80°C.

After incubation for 30 minutes the residual activity was measured usingthe Phadebas assay (described in the “Materials and Method” secionabove. The residual activity in the samples was measured using BrittonRobinson buffer pH 7.3. The decline in residual activity was measuredrelative to a corresponding reference solution of the same enzyme at 0minutes, which has not been incubated at high pH and 80° C.

The percentage of the initial activity as a function is shown in thetable below for the parent enzyme (SEQ ID NO: 2) and for the variants inquestion. Variant Residual activity after 30 min Δ(D183-G184) 4%Δ(D183-G184) + P459T 25% Δ(D183-G184) + K458R 31% Δ(D183-G184) + K311R10%

Example 8 Determination of Calcium Stability at Alkaline pH of Variantsof the Parent α-Amylase having the Amino Acid Sequence Shown in SEQ IDNO: 1, SEQ ID NO: 2 and SEQ ID NO: 4.

A: Calcium Stability of Variants of the Sequence in SEQ ID NO: 1

The measurement were made using solutions of the respective variants in100 mM CAPS buffer adjusted to pH 10.5 to which polyphosphate was added(at time t=0) to give a final concentration of 2400 ppm. The solutionswere incubated at 50° C. After incubation for 20 and 30 minutes theresidual activity was measured using the PNP-G7 assay (described above).The residual activity in the samples was measured using Britton Robinsonbuffer pH 7.3. The decline in residual activity was measured relative toa corresponding reference solution of the same enzyme at 0 minutes,which has not been incubated at high pH and 50° C.

The percentage of the initial activity as a function is shown in thetable below for the parent enzyme (SEQ ID NO: 1) and for the variants inquestion. Residual activity Residual activity Variant after 20 min after30 min Δ(T183-G184) 32% 19% Δ(T183-G184) + A186T 36% 23% Δ(T183-G184) +K185R + A186T 45% 29% Δ(T183-G184) + A186I 35% 20% Δ(T183-G184) + N195F44% n.d.n.d. = Not determinatedB: Calcium Stability of Variants of the Sequence in SEQ ID NO: 2

In this serie of analysis purified samples of enzymes were used. Themeasurement were made using solutions of the respective variants in 100mM CAPS buffer adjusted to pH 10.5 to which polyphosphate was added (attime t=0) to give a final concentration of 2400 ppm. The solutions wereincubated at 50° C.

After incubation for 20 and 30 minutes the residual activity wasmeasured using the PNP-G7 assay (described above). The residual activityin the samples was measured using Britton Robinson buffer pH 7.3. Thedecline in residual activity was measured relative to a correspondingreference solution of the same enzyme at 0 minutes, which has not beenincubated at high pH and 50° C.

The percentage of the initial activity as a function is shown in thetable below for the parent enzyme (SEQ ID NO: 2) and for the variants inquestion. Residual activity Residual activity Variant after 20 min after30 min Δ(D183-G184) + M323L 21% 13% Δ(D183-G184) + M323L + R181S 32% 19%Δ(D183-G184) + M323L + A186T 28% 17% Δ(D183-G184) + M323L + 30% 18%A186R Δ(D183-G184) 30% 20% Δ(D183-G184) + N195F 55% 44%

In this serie of analysis culture supernatants were used. Themeasurement were made using solutions of the respective variants in 100mM CAPS buffer adjusted to pH 10.5 to which polyphosphate was added (attime t=0) to give a final concentration of 2400 ppm. The solutions wereincubated at 50° C.

After incubation for 30 minutes the residual activity was measured usingthe Phadebas assay as described above. The residual activity in thesamples was measured using Britton Robinson buffer pH 7.3. The declinein residual activity was measured relative to a corresponding referencesolution of the same enzyme at 0 minutes, which has not been incubatedat high pH and 50° C.

The percentage of the initial activity as a function is shown in thetable below for the parent enzyme (SEQ ID NO: 2) and for the variants inquestion. Variant Residual activity after 30 min Δ(D183-G184) 0%Δ(D183-G184) + P459T 19% Δ(D183-G184) + K458R 18% Δ(D183-G184) + T461P13% Δ(D183-G184) + E346Q + K385R 4%C: Calcium Stability of Variants of the Sequence in SEQ ID NO: 4

The measurement were made using solutions of the respective variants in100 mM CAPS buffer adjusted to pH 10.5 to which polyphosphate was added(at time t=0) to give a final concentration of 2400 ppm. The solutionswere incubated at 60° C. for 20 minutes.

After incubation for 20 minutes the residual activity was measured usingthe PNP-G7 assay (described above). The residual activity in the sampleswas measured using Britton Robinson buffer pH 7.3. The decline inresidual activity was measured relative to a corresponding referencesolution of the same enzyme at 0 minutes, which has not been incubatedat high pH and 60° C.

The percentage of the initial activity as a function is shown in thetable below for the parent enzyme (SEQ ID NO: 4) and for the variants inquestion. Residual activity after Variant 20 min Termamyl (SEQ ID NO: 4)17% N265R 28% N265D 25%

Example 9 Activity Measurement at Medium Temperature of α-Amylaseshaving the Amino Acid Sequence Shown in SEQ ID NO: 1.

A: α-Amylase Activity of Variants of the Sequence in SEQ ID NO: 1

The measurement were made using solutions of the respective variants in50 mM Britton Robinson buffer adjusted to pH 7.3 and using the Phadebasassay described above. The activity in the samples was measured at 37°C. using 50 mM Britton Robinson buffer pH 7.3 and at 25° C. using 50 mMCAPS buffer pH 10.5.

The temperature dependent activity and the percentage of the activity at25° C. relative to the activity at 37° C. is shown in the table belowfor the parent enzyme (SEQ ID NO: 1) and for the variants in question.NU(25° C.)/ Variant NU/mg 25° C. NU/mg 37° C. NU(37° C.) SP690 144035000 4.1% Δ(T183-G184) 2900 40000 7.3% Δ(T183-G184) + K269S 1860 1200015.5% Δ(Q174) 3830 38000 7.9%

Another measurement was made using solutions of the respective variantsin 50 mM Britton Robinson buffer adjusted to pH 7.3 and using thePhadebas assay described above. The activity in the samples was measuredat 37° C. and 50° C. using 50 mM Britton Robinson buffer pH 7.3.

The temperature dependent activity and the percentage of the activity at37° C. relative to the activity at 50° C. is shown in the table belowfor the parent enzyme (SEQ ID NO: 1) and for the variants in question.NU(37° C.)/ Variant NU/mg 37° C. NU/mg 50° C. NU(50° C.) SP690 (seq IDNO: 1) 13090 21669 60% K269Q 7804 10063 78%B: α-Amylase Activity of Variants of the Sequence in SEQ ID NO: 2

The measurement were made using solutions of the respective variants in50 mM Britton Robinson buffer adjusted to pH 7.3 and using the Phadebasassay described above. The activity in the samples was measured at both25° C. and 37° C. using 50 mM Britton Robinson buffer pH 7.3.

The temperature dependent activity and the percentage of the activity at25° C. relative to the activity at 37° C. is shown in the table belowfor the parent enzyme (SEQ ID NO: 2) and for the variants in question.NU/mg NU/mg NU(25° C.)/ Variant 25° C. 37° C. NU(37° C.) Δ(D183-G184) +M323L 3049 10202 30% Δ(D183-G184) + M323L + R181S 18695 36436 51%C: α-Amylase Activity of Variants of the Sequence in SEQ ID NO: 4

The measurement were made using solutions of the respective variants in50 mM Britton Robinson buffer adjusted to pH 7.3 and using the Phadebasassay described above. The activity in the samples was measured at both37° C. using 50 mM Britton Robinson buffer pH 7.3 and at 60° C. using 50mM CAPS buffer pH 10.5.

The temperature dependent activity and the percentage of the activity at37° C. relative to the activity at 60° C. is shown in the table belowfor the parent enzyme (SEQ ID NO: 4) and for the variants in question.Variant NU/mg 37° C. NU/mg 60° C. NU(37° C.)/NU(60° C.) Termamyl  74004350 170% Q264S 10000 4650 215%

Example 10

Construction of Variants of Parent Hybrid BAN:1-300/Termamyl:301-483α-amylase

Plasmid pTVB191 contains the gene encoding hybrid α-amylaseBAN:1-300/Termamyl:301-483 as well as an origin of replicationfunctional in Bacillus subtilis and the cat gene conferringchloramphenicol resistance.

Variant BM4 (F290E) was constructed using the megaprimer approach(Sarkar and Sommer, 1990) with plasmid pTVB191 as template.

Primer p1 (SEQ ID NO: 52) and mutagenic oligonucleotide bm4 (SEQ ID NO:47) were used to amplify a 444 bp fragment with polymerase chainreaction (PCR) under standard conditions. This fragment was purifiedfrom an agarose gel and used as ‘Megaprimer’ in a second PCR with primerp2 (SEQ ID NO: 53) resulting in a 531 bp fragment. This fragment wasdigested with restriction endonucleases HinDIII and Tth111I. The 389 bpfragment produced by this was ligated into plasmid pTVB191 that had beencleaved with the same two enzymes. The resulting plasmid was transformedinto B. subtilis SHA273. Chloramphenicol resistant clones were selectedby growing the transformants on plates containing chloramphenicol aswell as insoluble starch. Clones expressing an active α-amylase wereisolated by selecting clones that formed halos after staining the plateswith iodine vapour. The identity of the introduced mutations wasconfirmed by DNA sequencing.

Variants BM5(F290K), BM6(F290A), BM8(Q360E) and BM11(N102D) wereconstructed in a similar way. Details of their construction are givenbelow.

Variant: BM5(F290K)

-   mutagenic oligonucleotide: bm5 (SEQ ID NO: 48)-   Primer (1st PCR): p1 (SEQ ID NO: 52)-   Size of resulting fragment: 444 bp-   Primer (2nd PCR): p2 (SEQ ID NO: 53)-   Restriction endonucleases: HinDIII, Tth111I-   Size of cleaved fragment: 389 bp    Variant: BM6(F290A)-   mutagenic oligonucleotide: bm6 (SEQ ID NO: 49)-   Primer (1st PCR): p1 (SEQ ID NO: 52)-   Size of resulting fragment: 444 bp-   Primer (2nd PCR): p2 (SEQ ID NO: 53)-   Restriction endonucleases: HinDIII, Tth111I-   Size of cleaved fragment: 389 bp    Variant: BM8(Q360E)-   mutagenic oligonucleotide: bm8 (SEQ ID NO: 50)-   Primer (1st PCR): p1 (SEQ ID NO: 52)-   Size of resulting fragment: 230 bp-   Primer (2nd PCR): p2 (SEQ ID NO: 53)-   Restriction endonucleases: HinDIII, Tth111I-   Size of cleaved fragment: 389 bp    Variant: BM11(N102D)-   mutagenic oligonucleotide: bmll (SEQ ID NO: 51)-   Primer (1st PCR): p3 (SEQ ID NO: 54)-   Size of resulting fragment: 577-   Primer (2nd PCR): p4 (SEQ ID NO: 55)-   Restriction endonucleases: HinDIII, PvuI-   Size of cleaved fragment: 576

Mutagenic Oligonucleotides: bm4: (SEQ ID NO: 47) F290E primer 5′ GTG TTTGAC GTC CCG CTT CAT GAG AAT TTA CAG G bm5: (SEQ ID NO: 48) F290K primer5′ GTG TTT GAC GTC CCG CTT CAT AAG AAT TTA CAG G bm6: (SEQ ID NO: 49)F290A primer 5′ GTG TTT GAC GTC CCG CTT CAT GCC AAT TTA CAG G bm8: (SEQID NO: 50) Q360E primer 5′ AGG GAA TCC GGA TAG CCT GAG GTT TTC TAC GGbm11: (SEQ ID NO: 51) N102D primer 5′ GAT GTG GTT TTG GAT CAT AAG GCCGGC GCT GAT G

Other Primers: p1: 5′ CTG TTA TTA ATG CCG CCA AAC C (SEQ ID NO: 52) p2:5′ G GAA AAG AAA TGT TTA CGG TTG (SEQ ID NO: 53) CG p3: 5′ G AAA TGA AGCGGA ACA TCA AAC (SEQ ID NO: 54) ACG p4: 5′ GTA TGA TTT AGG AGA ATT CC(SEQ ID NO: 55)

Example 11 α-Amylase Activity at Alkaline pH of Variants of ParentBAN:1-300/Termamyl:301-483 Hybrid α-Amylase.

The measurements were made using solutions for the respective enzymesand utilizing the Phadebas assay (described above). The activity wasmeasured after incubating for 15 minutes at 30° C. in 50 mMBritton-Robinson buffer adjusted to the indicated pH by NaOH. NU/mgenzyme pH wt Q360E F290A F290K F290E N102D 8.0 5300 7800 8300 4200 66006200 9.0 1600 2700 3400 2100 1900 1900

REFERENCES CITED

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1-39. (canceled)
 40. A variant alpha-amylase, which variant has□-amylase activity, has at least 90% homology with the amino acidsequence shown in SEQ ID. NO. 3, and which comprises one or more of thefollowing substitutions (using SEQ ID NO: 2 for numbering): (a)R181A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,or V; (b)G182A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,or V; (c)D183A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,or V; (d)G184A,R,D,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y, or V; (e)K185A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,or V; and (f)A186D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,or V.
 41. A variantalpha-amylase, which variant has alpha-amylase activity, has at least95% homology with the amino acid sequence shown in SEQ ID. NO. 3, andwhich comprises one or more of the following substitutions (using SEQ IDNO: 2 for numbering): (a) R181A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y, or V;(b) G182A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y, or V; (c)D183A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y, or V; (d)G184A,R,D,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y, or V; (e)K185A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y, or V; and (f)A186D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y, or V.
 42. The variant of claim40, wherein the variant comprises a substitution ofR181A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y, or V.
 43. The variant of claim40, wherein the variant comprises a substitution ofG182A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y, or V.
 44. The variant of claim40, wherein the variant comprises a substitution ofD183A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y, or V.
 45. The variant of claim40, wherein the variant comprises a substitution ofG184A,R,D,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y, or V.
 46. The variant of claim40, wherein the variant comprises a substitution ofK185A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y, or V.
 47. The variant of claim40, wherein the variant comprises a substitution ofA186D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y, or V.
 48. The variant of claim41, wherein the variant comprises a substitution ofR181A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y, or V.
 49. The variant of claim41, wherein the variant comprises a substitution ofG182A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y, or V.
 50. The variant of claim41, wherein the variant comprises a substitution ofD183A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y, or V.
 51. The variant of claim41, wherein the variant comprises a substitution ofG184A,R,D,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y, or V.
 52. The variant of claim41, wherein the variant comprises a substitution ofK185A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y, or V.
 53. The variant of claim41, wherein the variant comprises a substitution ofA186D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y, or V.
 54. A DNA constructcomprising a DNA sequence encoding an alpha-amylase variant according toclaim
 40. 55. A recombinant expression vector which carries a DNAconstruct according to claim
 54. 56. A cell which is transformed with aDNA construct according to claim
 54. 57. A DNA construct comprising aDNA sequence encoding an alpha-amylase variant according to claim 41.58. A recombinant expression vector which carries a DNA constructaccording to claim
 57. 59. A recombinant expression vector which carriesa DNA construct according to claim 57.